The proposed research project will continue to kinetically evaluate two important post-translational events which occur during the formation of human hemoglobin in vitro and in vivo. One essential process involves the proper assembly of the hemoglobin tetramer from either nascent globin or heme-containing polypeptide alpha and beta chains. Recent biochemical and hematological findings support the premise that the rate of assembly (an electrostatic process) may be an important determinant of hemoglobin distribution in the circulating erythrocytes of normal individuals and those with hematological disorders, including thalassemia and heme deficiency. The rates of subunit assembly of normal and distinctly charged variant human hemoglobins from heme-containing oxy-alpha and non-alpha-subunits will be spectrophotometrically monitored by stopped flow techniques developed in the laboratory. The rate of combination of heme-containing and heme-free globin chains (another probable pathway of assembly) will be evaluated using fluorescent quenching rapid kinetic techniques. The stability of apoglobin, another possible subunit assembly intermediate will be investigated by monitoring the slow transfer of a heme-containing subunit into apoglobin with time by either electrophoresis or high performance liquid chromatography (HPLC). Another equally vital post-synthetic step involves proper insertion of the Fe-protoporphyrin IX group into possible globin subunit assembly intermediates. Proper incorporation of the heme prosthetic group is essential to the formation of an oxygen transporting hemoglobin protein. Of particular interest is kinetically evaluating the rate of heme insertion into two globin species found in vivo: apoglobin and seni-alpha-hemoglobin, and can be accomplished by Soret spectral monitoring in a stopped flow apparatus. Studies suggest that the alpha- and beta- heme pockets are not equivalent and chain heterogeneity of hene binding will be evaluated. Finally, the role of the red cell membrane (in particular the cytoplassic domain of band 3; CDB3) in the assembly and stability of heme containing subunit assembly intermediates will be evaluated. This CDB3-hemoglobin binding event which may be governed by electrostatic forces (i.e. identical to that proposed for that of subunit assembly) will be investigated by fluorescence techniques.